In recent years, in food manufacturing sites, clinical sites, environments for protecting cultural assets, it has become important to check the presence of microorganisms such as fungi to confirm safety, as well as to prevent the proliferation thereof.
In such inspection of fungi, in general, a morphology observation method (cultivation method) is generally conducted in which a sample is collected from the environment and then pre-cultivated, and then, after incubation of about 20 days in a culture medium which is optimum for the type of fungi, morphological features are observed, whereby fungi are identified (see Patent Document 1).
However, in this method, since cultivation is required to be conducted separately according to the type of fungi, the inspection process becomes complicated. In addition, since cultivation takes a long period of time, the cultivation method is not appropriate for inspection which requires promptness, such as detection indoors where people stay and inspection of foods, for example. Further, identification cannot be made unless fungi spore that shows morphological features of fungi is formed, resulting in wasting of time and labor.
Recently, in inspection of fungi, detection by a gene has been conducted. Specifically, after cultivating a sample collected from an environment, DNA is extracted from a cultivated cell, a target region is amplified by a PCR (polymerase chain reaction) method, and an amplified product is analyzed, thereby to identify fungi present in the sample. As a method for analyzing an amplified product, for example, a method in which the size of an amplified product is analyzed by electrophoresis or fungi present in a sample is identified by means of a DNA chip, to which a probe which connects complimentarily to an amplified product is fixed, or the like have been proposed (see Patent Documents 2 to 6).
Patent Document 1: JP-A-2007-195454
Patent Document 2: JP-A-2008-35773
Patent Document 3: JP-A-2008-278848
Patent Document 4: JP-A-2008-278861
Patent Document 5: JP-A-2010-4879
Patent Document 6: JP-A-2009-284832
However, even by such an identification method using genes, when the type of fungi are very similar, it is significantly difficult to identify the type of fungi only by the presence or absence of a single target gene or the size thereof. For example, this method is not suited to an inspection where the identification accuracy on the level of type is required.
In Patent Documents 2 to 4, identification is conducted by using the ITS region (Internal Transcribed Spacer) in the gene of various fungi as a region to be amplified. By identification of fungi based on the ITS region, a false positive reaction tends to occur more frequently as the number of type of fungi is increased, thereby lowering the accuracy of the inspection.
On the other hand, Patent Documents 5 and 6 each disclose identification by using the β-tubulin gene as a region to be amplified. In these documents, it is stated that a specific type of fungi can be detected by using the β-tubulin gene as a region for amplification.
However, if the β-tubulin gene alone is used as a region for amplification, as in the case where only the ITS region is used as a region for amplification, a false positive reaction may occur.